**2.3. Sample preparation for Stable Isotopes Analysis (SIA)**

80 New Approaches to the Study of Marine Mammals

also explained in Figure 1.

Galapagos sea lion pups as indicators

Diet

their mothers because*δ*15*N* isotopic enrichment.

**2.2. Fish collection and homogenization** 

biomagnification

of POPs

study design to justify the use of pups as ecosystem based sentinels of biomagnification is

Pups-mother (milk)

Predator─Prey relationship

(detritus)

Diet

**Thread herring (planktivorous) Mullet (detritivorous)**

Diet (POPs)

POPs

Water/Plankton Sediment

POPs

**Figure 1.** Conceptual model illustrating the bioaccumulation process in a representative, food chain of the Galapagos sea lion. Piscivorous Galapagos sea lions can be exposed to persistent organic pollutants (POPs), mainly through dietary ingestion. Low trophic level prey fish can absorb POPs from water and plankton (planktivorous fish), as well as from sediments (detritivorous fish). Nursing pups can bioaccumulate POPs from adult females by nursing and thus occupy a higher tropic level relative to

Two species of fish (mullets, *Mugil curema*; *n* = 11; and, Galapagos thread herrings, *Ophistonema berlangai*; *n* = 4), which for the purpose of this study were assumed to be major prey items of Galapagos sea lions, were collected from Galapagos waters by fishers during Each set of hair samples collected from Galapagos sea lion pups was cleaned for lipid and particle removal by washing the hair three times with a chloroform:methanol 2:1 v/v solution using a clean Pasteur glass pipette. Samples were transferred into labelled scintillation vials and desiccated overnight, and, then, lyophilized using a freeze drier (Free Zone ® Plus 4.5 Liter Cascade; Labconco, Kansas City, MO) for 24 hr (Vacuum pressure set point: 0.01 mBar).

Fish biopsy samples were freeze dried overnight (Vacuum pressure set point: 0.01 mBar). Biopsy samples were weighed and freeze dried again to determine if there were differences in weights after the second freeze drying. Once the sample weight was constant (i.e., no remaining moisture), one set of freeze dried samples was stored in the desiccator until further analysis for *δ*15*N*. The set of freeze dried replicates underwent an extraction protocol to remove lipids to be used for *δ*13*C* analysis. First, freeze dried samples were pulverized using a mortar and transferred into a glass tube for lipid extraction by adding 5ml of chloroform:methanol 2:1 v/v; and, then vortex mixed for 30 seconds. Solids were dispersed with sonification in bath sonicator for 10 min. Samples were allowed to settle for 30 min at room temperature, followed by an additional 30 second vortex and sonification. Samples were centrifuged for 5 minutes at 1000 rpm (model GS6R, Beckman, USA) to enhance pellet formation. The solvent was carefully removed with glass Pasteur pipette (pipette was changed for each sample), without transferring any particulate matter, and the solvent was disposed in the waste bottle. A second extraction was repeated. The supernatant was carefully removed with pipette and the residue was left at -20ºC overnight. Samples were dried under Nitrogen and transferred to a clean, amber vial for analysis of stable isotopes of carbon and nitrogen.

#### **2.4. Stable Isotopes Analysis (SIA)**

Carbon and nitrogen isotopic analyses on fish biopsies and Galapagos sea lion hair were accomplished by continuous flow, isotopic ratio mass spectrometry (CF-IRMS) using a GV-Instruments® IsoPrime attached to a peripheral, temperature-controlled, EuroVector® elemental analyzer (EA) (University of Winnipeg Isotope Laboratory, UWIL). One-mg samples were loaded into tin capsules and placed in the EA auto-sampler along with internally calibrated carbon/nitrogen standards. Nitrogen and carbon isotope results are expressed using standard delta (*δ*) notation in units of per mil (‰).The delta values of carbon (*δ*13*C*) and nitrogen (*δ*15*N*) represent deviations from a standard. *δ*15*N* isotope ratios (‰) were determined using the following equation [21,26]:

Assessing Biomagnification and Trophic Transport of Persistent Organic Pollutants in the Food Chain of the Galapagos Sea Lion (*Zalophus wollebaeki*): Conservation and Management Implications 83

Although pups instead of adult individual sea lions were sampled in this study, the *δ*15*N* signature in the pup is expected to reflect the isotopic nitrogen signature of the mother, as pups feed only on mothers' tissue (i.e., milk proteins) analogous to a predator-prey relationship, resulting in a *δ*15*N* isototipc enrichment of 2.1‰ and 0.9‰ *δ*13*C* enrichment in relation to adult females [41, 42]. Because of lactation, pups can be at a higher trophic level than their mothers (Figure 1). However, the *δ*15*N* signature in the pups can provide useful

Contaminant analyses were conducted in the Regional Dioxin Laboratory (RDL) at the Institute of Ocean Sciences (IOS), Fisheries and Ocean Canada (DFO), based on analytical methodologies described elsewhere [44]. In brief, the muscle-blubber biopsy samples of Galapagos sea lion pups (0.053 to 0.212 g wet weight) and subsamples of fish homogenate (9.23 to 10.5 g) were spiked with a mixture of surrogate internal standards which contained all fifteen 13C12-labeled PCBs, and a mixture of labelled organochlorine pesticides (OCPs): D3 1,2,4-Trichlorobenzene, 13C6 1,2,3,4 Tetrachlorobenzene, 13C6 Hexachlorobenzene, 13C6*�*-HCH, 13C6*�*-HCH, 13C10 trans Nonachlor, 13C12 TeCB-47, 13C12*p*,*p*'-DDE, 13C12 Dieldrin, 13C12*o*,*p*-DDD, 13C12*p*,*p*'-DDD, 13C12*o*,*p*-DDT, 13C12*p*,*p*'-DDT, 13C10 Mirex. All surrogate internal standards were purchased from Cambridge Isotope Laboratories (Andover, MA). The spiked samples were homogenized with Na2SO4 in a mortar, transferred quantitatively into an extraction column, and extracted with DCM/hexane (1:1 v/v). For some of the samples the extract formed two layers/phases, a waxy-precipitate layer and the solvent layer. The solvent layer was transferred to a clean flask and the waxy precipitate was treated with several aliquots of hexane and DCM. Each of these was transferred to the flask that contained the solvent layer of the extract. Despite the treatment with additional volumes of hexane and DCM, vortexing and pulverization, the waxy precipitate (for sea lions) did not dissolved in the solvents used and as a result it was not included in the corresponding sample extract that was used for lipid and contaminants determinations.The DCM:Hexane sample extracts were evaporated to dryness and the residue was weighted in order to determine the total lipid in the samples. Subsequently the residue was re-suspended in 1:1 DCM/Hexane and divided quantitatively into two aliquots. The larger aliquot (75% of the extract) was subjected to sample-cleanup for PCBs determinations. The remaining (25% of the extract) was used for

Sample extracts were analyzed for PCB congeners and target OCPs by gas chromatography/high-resolution mass spectrometry (GC/HRMS). The specific methodology and protocols for the quantification and analytical methods to determine PCB congeners

and OCPs have previously been reported in prior published papers (34, 35).

information about the foraging habits (i.e., diet) of adult female animals [43].

**2.6. Sample preparation for chemical analysis** 

OCP determinations.

**2.7. PCB and OC pesticides analyses** 

$$\delta^{15}\text{N} = \left[ \{ ^{15}\text{N} / ^{14}\text{N}\_{\text{SAMPLE}} / ^{15}\text{N} / ^{14}\text{N}\_{\text{STANDARD}} \} - 1 \right] \ge 1000 \text{ J}$$

where 15*N*/14*N*SAMPLE is the isotope ratio of the tissue sample analyzed; and, 15*N*/14*N*STANDARD represents the ratio of the international standard of atmospheric *N*2 (air), IAEA-N-1 (IAEA, Vienna), for *δ*15*N*. The equivalent equation for *δ*13*C* isotope ratios (‰) is:

$$\delta^{13}\mathcal{C} = \left[ \{ ^{13}\text{C}/^{12}\text{C}\_{\text{SAMPLE}}/^{13}\text{C}/^{12}\text{C}\_{\text{STANDARD}} \} - 1 \right] \ge 1000 \text{ } \delta$$

The standard used for carbon isotopic analyses was the Vienna PeeDee Belemnite (VPDB). Analytical precision, determined from the analysis of duplicate samples, was ±0.13‰ for *δ*13*C* and ±0.6‰ for *δ*15*N*. The analytical precision based on standards, which are more isotopically homogeneous than samples, was ± 0.19‰ for *δ*13*C* and ±0.24 for *δ*15*N*.

#### **2.5. Trophic level estimations**

The trophic positions (TPCONSUMER) of the prey species (i.e. fish) and the predator (Galapagos sea lion) were determined relative to the baseline *δ*15*N* (assumed to occupy a trophic level 2), using the following algorithm [37, 38]:

$$\text{TP}\_{\text{CONSILHER}} = \frac{\left(\ $^{15}\text{N}\_{\text{CONSILPER}} \cdot \$ ^{15}\text{N}\_{\text{BASEINE}}\right)}{3.4} + 2$$

Where *δ*15*N*CONSUMER is the average *δ*15*N* signature value of the predator; *δ*15*N*BASELINE is the *δ*15*N* signature at the base of the food web; and 3.4‰ is the isotopic, trophic level enrichment factor (*∆*15*N*), recommended to be used for constructing food webs when a priori knowledge of *∆*15*N* is unavailable [39]. The *δ*15*N*BASELINE was established as the *δ*15*N* signature of the particulate organic matter (POM) of bottom sediments in the eastern equatorial Pacific Ocean (250 km south of the islands) with a value of 5.5‰ [31, 40], which is relatively close to the *δ*15*N* value of 7.3‰, reported recently for phytoplankton in the Galapagos [30]. The rationale for using this signature is supported by the fact that the assimilation of nitrogen (i.e., NO3¯) up taken from near surface marine waters by phytoplankton is reflected by *δ*15*N* values of POM, which is also a major component of the carbon flux and sediments [40].

Although pups instead of adult individual sea lions were sampled in this study, the *δ*15*N* signature in the pup is expected to reflect the isotopic nitrogen signature of the mother, as pups feed only on mothers' tissue (i.e., milk proteins) analogous to a predator-prey relationship, resulting in a *δ*15*N* isototipc enrichment of 2.1‰ and 0.9‰ *δ*13*C* enrichment in relation to adult females [41, 42]. Because of lactation, pups can be at a higher trophic level than their mothers (Figure 1). However, the *δ*15*N* signature in the pups can provide useful information about the foraging habits (i.e., diet) of adult female animals [43].
